The blocking reagents which is used when a gene is detected by using a nylon membrane or a nitrocellulose membrane, include Denhalt reagent (50× Denhalt solution: 1% (w/v) Ficoll, 1% (w/v) polyvinyl pyrrolidone, 1% (w/v) bovine serum albumin), heparin, and skim milk. However, even by use of these blocking reagents, a blocking effect is not satisfactory, and sometimes a reproducible result cannot be obtained.
In the case where a slide glass is used as a substrate, a method is reported in which the slide glass coated with poly-L-lysine is blocked with succinic anhydride (P. O. Brown et al., Genome Res, 1996; 6: 639–645; Japanese Patent Laid-open Publication No. H11-514872). This method tries to delete a charge of an amino group by coupling the amino group on the slide glass with succinic anhydride. However, this method has various problems other than the blocking of the amino group.
For example, since the slide glass is immersed in a solution during blocking, DNA which was spotted to the slide glass was washed off during blocking, resulting in nonspecific adsorption, not good appearance, and no reproducibility, or the like. Thus, reliability of obtained data was insufficient.
In the case where a DNA chip is utilized, a fluorescence labeled-compound is often used for detection. Since such labeled compounds usually have a charge (since fluorescent dyes contain a sulfone group (—SO3H) in many cases), DNA labeled with these compounds is apt to be nonspecifically adsorbed via ionic bond.
As to the method for labeling DNA, such methods are often used that DNA is labeled from RNA by incorporating a labeled dUTP with a reverse transcription reaction; or that it is labeled from DNA of genome by incorporating a labeled dUTP with a PCR reaction or the like. Although these probes are purified by ethanol precipitation after the reaction, unreacted dye labeled dUTP is often unpurified and left. These unpurified substance often causes nonspecific adsorption.